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REVIEW: RNA Integrity and the Effect on the Real-Time Qrt-PCR Performance

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REVIEW: RNA Integrity and the Effect on the Real-Time Qrt-PCR Performance Molecular Aspects of Medicine 27 (2006) 126–139 www.elsevier.com/locate/mam Review RNA integrity and the effect on the real-time qRT-PCR performance Simone Fleige a, Michael W. Pfaffl a,b,* a Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University of Munich, 85350 Freising, Germany b TATAA Biocenter Germany, Freising-Weihenstephan, Germany Abstract The assessment of RNA integrity is a critical first step in obtaining meaningful gene expres- sion data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern mole- cular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality now- adays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as * Corresponding author. Present address: Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University of Munich, 85350 Freising, Germany. Tel.: +49 8161 71 3511; fax: +49 8161 71 4204. E-mail address: michael.pfaffl@wzw.tum.de (M.W. Pfaffl). 0098-2997/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2005.12.003 S. Fleige, M.W. Pfaffl / Molecular Aspects of Medicine 27 (2006) 126–139 127 good total RNA quality and higher than eight as perfect total RNA for downstream application. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: RNA quality; RNA quantity; qRT-PCR; 2100 Bioanalyzer; Experion; Lab-on-chip Contents 1. The particular importance of RNA quality . 127 1.1. Introduction ................................................ 127 1.2. RNA extraction . ............................................ 128 1.3. RNA quantity and quality assessment . ........................... 129 2. Integrity of RNA and its effect on real-time qRT-PCR . 133 2.1. General aspects. ............................................ 133 2.2. Effect on the mRNA quantification . ........................... 133 2.3. Association between RNA quality and length of the amplified product . 136 3. Conclusion . 137 References . 137 1. The particular importance of RNA quality 1.1. Introduction The accuracy of gene expression evaluation is recognised to be influenced by the quantity and quality of starting RNA. Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labour-intensive, time-consuming and highly expensive (Raeymarkers, 1993; Imbeaud et al., 2005). It is preferable to use high-quality intact RNA as a starting point in molecular biological as well as in diagnostic applications. Especially in quantitative RT-PCR, micro-arrays, ribonuclease-protection-assay, in situ hybrid- ization, northern blot analysis, RNA mapping, in vitro translation, cDNA library construction and any kind of array applications the integrity of the used total RNA should be checked. Especially in clinical application with unique or limited tis- sue material, e.g. obtained after surgery, a reliable RNA quality analysis is necessary (Bustin and Nolan, 2004b). As a consequence, several steps during tissue handling have to be carefully controlled in order to preserve the quality and integrity of the RNA material. It is well known that RNA is sensitive to degradation by postmortem processes and inadequate sample handling or storage (Perez-Novo et al., 2005). Thus the competency to quickly assess RNA quality using minor amounts has become increasingly important as the following measures of mRNA transcripts have become more expensive and more comprehensive. 128 S. Fleige, M.W. Pfaffl / Molecular Aspects of Medicine 27 (2006) 126–139 1.2. RNA extraction The quality and quality of purified RNA is variable and after the extraction dur- ing long storage rather unstable (Bustin et al., 2005). Especially long mRNA frag- ments up to 10 kb are very sensitive to degradation. This can happen through cleavage of RNAses introduced by handling with RNA samples. The most obvious problem concerns the degradation of the RNA and this is best addressed by insisting that every RNA preparation is rigorously assessed for quality and quantity. The extraction and purification procedure of total RNA must fulfill the following criteria (Bustin and Nolan, 2004b; Pfaffl, 2005a): • free of protein (absorbance 260 nm/280 nm); • free of genomic DNA; • should be undegraded (28S:18S ratio should be roughly between 1.8 and 2.0, with low amount of short fragments); • free of enzymatic inhibitors for RT and PCR reaction, which is strongly depen- dent on the purification and clean-up methods; • free of any substances which complex essential reaction co-factors, like Mg2+ or Mn2+; • free of nucleases for extended storage; There are a substantial quantity of problems that affect reproducibility, and hence the relevance of results. The source of RNA, sampling techniques (biopsy material, single cell sampling, laser micro-dissection) as well as RNA isolation techniques (either total RNA or poly-adenylated RNA isolation techniques) often vary signifi- cantly between processing laboratories (Bustin and Nolan, 2004b; Pfaffl, 2004). The RNA quality can be different between two extraction methods, e.g. performed by hand or by an automatic extraction system. The isolated total cellular RNA with the liquid extraction, e.g. Trizol (Roche Diagnostics, Germany) or TriFast (peqlab, Germany), has different RNA quality, whereas only the type of homogenization is changed (Fleige and Pfaffl, 2006). Due to its inherent susceptibility to ubiquitous RNases and its chemical instability, RNA is readily endangered by base- or enzyme-catalyzed hydrolysis. Researchers must take into account a variety of factors, which influence their ability to obtain high-quality RNA that is free of con- tamination such as RNases, proteins and genomic DNA. These factors include yield variations, processing requirements, and sample availability of different cells or tissues. The best RNA yield is obtained from tissue that has been diced into small fragments with a scalpel prior to being frozen by submerging in liquid nitrogen. The samples must be homogenized using a bead mill or a mechanical homogenizer (Bustin and Nolan, 2004b). Further problem may arise in the case of research on human or animal tissue sam- pling techniques and the time dependency until the tissue is stored safely in RNase inhibitors or RNA-later (Ambion, USA). It is often very challenging to decrease this sampling time to a minimum within the framework of clinical routine procedures, or in animal experiments during a slaughtering process. The RNA quality may also be S. Fleige, M.W. Pfaffl / Molecular Aspects of Medicine 27 (2006) 126–139 129 impaired in samples stored for a long time or under sub-optimal conditions (Schoor et al., 2003). 1.3. RNA quantity and quality assessment Conventional methods are often not sensitive enough, not specific for single- stranded RNA, and disposed to interferences from contaminants present in the sample (Imbeaud et al., 2005). The assessment of RNA integrity can do by various methods: the classical gel OD measurement, modern OD measurement via Nano- Drop, old fashioned denaturating agarose gel-electrophoresis or with high innova- tive lab-on-chip technologies like Bioanalyzer 2100 (Agilent Technologies, USA) and Experion (Bio-Rad Laboratories, USA). Quantity and quality assessment using a UV/VIS spectrophotometer should be performed at multiple wave lengths at 240 nm (background absorption and possible contaminations), 260 nm (specific for nucleic acids), 280 nm (specific for proteins), and 320 nm (background absorption and possible contaminations). On basis of the OD 260 the quantity, and by the ratio of the optical density (OD) of OD 260/280 the quality, OD 260/240 or OD 260/320 the purity and the extraction performance can be verified. An OD 260/280 ratio greater than 1.8 is usually considered an acceptable indicator of good RNA quality (Sambrook et al., 1989; Manchester, 1996). By the presence of genomic DNA the OD 260 measurement can compromised and leading to over-estimation of the actual and real RNA concentration. Further the used buffer and high salt concentrations will interfere with the result of the optical measurement and therefore the calculated RNA concentrations might be over- or under-estimated (own unpublished results). The accuracy of the OD 260/A280 method has been questioned, with a value
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